The Malaria Parasite Lifecycle and the Disease Pathogenesis
Malaria continues to place an unacceptable burden on the health and economic development in over 100 countries. One to two million people die of malaria each year and most of them are children under the age of five. A child dies from malaria every 30 seconds, and 350 to 500 million malaria cases are estimated annually worldwide (World Malaria Report 2008, “WHO/HTM/GMP/2008.1”, ISBN 978 92 4 156369 7).
Malaria is caused by the protozoan parasite of the genus Plasmodium. Four species of Plasmodium affect humans, and these are P. falciparum, P. vivax, P. ovale, and P. malariae. Of these, the P. falciparum is responsible for 90% of the world malaria mortality. In humans, the infection is initiated with the meal bite of an Anophilis mosquito injecting the sporozontes of the Plasmodium while feeding. After an initial incubation period in the human liver, the Plasmodium parasite undergoes an exoerythrocytic schizogony (i.e., an asexual replication), after which the merozoites (the daughters cells) enter the blood circulatory system and penetrate the red blood cells (RBCs) which become parasitized (pRBCs). In the pRBCs, the parasite grows by feeding off the hemoglobin; during this intra-RBC phase the parasite is known as a trophozoite. The early trophozoites are referred to as the ring form, due to their ring shape. In the late stage the trophozoites undergo a nuclear division, during which the parasite is known as a schizont. The trophozoites cycle terminates with schizonts undergoing an asexual replication, from which merozoites bud. Following the budding of the merozoites, the pRBC bursts, releasing into the blood stream these merozoites that quickly infect new RBCs. “Burst” is used herein to refer to the breakdown of the pRBC at the end of the parasite intra-erythrocyte lifecycle. The process continues cyclically with the number (percentage) of infected RBCs (parasitemia) growing rapidly and causing life-threatening consequences for the infected human. The intra-RBC cycle of the parasite varies between 24-72 hours, depending on the species, and the bursting of each pRBC releases several merozoites (Arrow et al., “Saving Life, Buying Time, Economics of Malaria Drugs in an Age of Resistance”, Institute of Medicine of the National Academies, 2004).
Out of the many merozoites infecting the RBCs, few differentiate into a sexual form known as microgametocytes. These larger parasites fill up the erythrocyte and are ingested by mosquitoes during the human blood feeding. These gametocytes reproduce inside the mosquito from which sporozoites are born. These sporozoites, born within the mosquito, are then transferred on to other humans during subsequent bites. The infection cycle from mosquito to human and back to mosquito and human again is thus completed. In order to reproduce, the malaria parasite requires a mosquito and a human phase. The disease would continue to perpetuate until the reproduction cycle is interrupted. This interruption could be achieved by either preventing the mosquito's bites or by eliminating the microgametocytes. In fact, while these microgametocytes are not responsible for the pathogenesis of the disease, they are directly responsible for the disease transmission and a key element in the reproduction cycle of the parasite (Miller et al., Nature 415: 673-679, 2002). Without the mosquito ingesting the gametocytes present in the individual's infected blood, the parasite would not reproduce in the mosquito and the infection cycle would be interrupted.
The parasite blood stage is responsible for the pathology associated with malaria. If untreated, malaria rapidly results in a life-threatening disease. The most common side effects include fever, anemia, and respiratory distress. As the number (percentage) of pRBCs increase, the patient progressively sickens. When the fraction of pRBCs reaches about 5% of the total RBCs, strokes, coma, and multiple organ failures are among the potential complications and causes of death (World Malaria Report 2008, “WHO/HTM/GMP/2008.1”, ISBN 978 92 4 156369 7 and Arrow et al., “Saving Life, Buying Time, Economics of Malaria Drugs in an Age of Resistance”, Institute of Medicine of the National Academies, 2004). This stage of the disease is referred to as severe malaria and cerebral malaria if the brain is the organ most affected by the disease. These stages of the disease lead to a rapid death if untreated in most cases. However, even under the current treatment method, the mortality rate of severe malaria is exceptionally high.
The malaria pathogenesis is a very complex process affecting several organs and tissues, and as of today it is not yet fully understood (Miller et al., Nature 415: 673-679, 2002). The principle pathophysiological future of severe malaria is the metabolic acidosis, which leads to the commonly observed respiratory distress in severe malaria. There are several causes of acidosis: i) Liver failure; ii) increase in lactic acid stimulated by the immune system (cytokines) that is reacting to the release of the parasite toxic byproduct (hemozoin) following the RBCs' burst; iii) the reduction of oxygen delivery to several tissues—the absence of oxygen in tissue further stimulates the formation of acidosis. The reduction of the oxygen delivery is caused by the combination of several factors. First, an increasing anemia due to the destruction of RBCs by the parasite—every 48 hours the pRBCs burst and are removed from the total RBC's count. Second, the P. falciparum alters the RBC's surface protein, causing the RBCs to change shape, deformability, ability to adhere to the vessel surface, and a reduction of the deformability in non-infected RBCs. These factors increase blood viscosity, obstruct the flow of other RBCs, and thus ultimately compromise the blood flow. The changeover in blood rheology that follows this set of events is ultimately the cause of malaria-induced complications, including kidney failures and strokes.
Current Malaria Treatments: Anti-Malarial Drugs and Exchange Transfusion.
At present, malaria is treated with one or more of numerous pharmaceutical drugs. A brief list of drugs includes chloroquine, sulfadoxine-pyrimethamine, mefloquine, atovaquone-proguanil, amodiaquine, quinine, doxycycline, and artemisin derivatives (Arrow et al., “Saving Life, Buying Time, Economics of Malaria Drugs in an Age of Resistance”, Institute of Medicine of the National Academies, 2004; Griffith et al., JAMA. 297(20):2264-2277, 2007; Yakoub et al., “Handbook of Drugs for Tropical Parasitic Infections”, Taylor & Francis Inc., 1995). In the last three decades, most malaria endemic regions have seen the growth of parasite strains that are immune to one or more drugs with only the exception of the artemisin compounds. However, the lack of artemisin resistance is believed to be due to the limited use of these compounds in endemic regions. In fact, the World Health Organization (WHO) fears that once the artemisin compounds are widely used, parasite strains would develop a resistance to the artemisin compounds and spread through the world endemic regions. If artemisin resistance were to develop, it would signal the end of anti-malarial drugs with severe consequence on the spread of the disease. The development of a parasite strain resistant to artemisin is highly probable. Research studies have shown that all drugs entered in the mass-market bore resistant parasite strains within ten years from their market introduction.
In addition to the growing parasite resistance, all anti-malarial drugs have other severe pitfalls and side effects. These drawbacks may include a low efficacy of a given anti-malarial that varies with the parasite strain or a considerable reduction in efficacy of a given anti-malarial regarding the progression of the disease into the severe stage. While a drug may effectively work in treating malaria in the early stage of the infection, its effectiveness is considerably lower in severe episodes. A further drawback to relying on anti-malarials is that a long term window for the drugs is often required before they become effective. Severe malaria strains may induce death within 24 hours, and since malaria infection often goes undetected and/or unrecognized, untreated malaria even in the early stage of the infection is a life threatening disease (World Malaria Report 2008, “WHO/HTM/GMP/2008.1”, ISBN 978 92 4 156369 7; Arrow et al., “Saving Life, Buying Time, Economics of Malaria Drugs in an Age of Resistance”, Institute of Medicine of the National Academies, 2004; Miller et al., Nature 415: 673-679, 2002; Griffith Kevin S. et al., JAMA. 297(20):2264-2277, 2007; Yakoub et al., “Handbook of Drugs for Tropical Parasitic Infections”, Taylor & Francis Inc., 1995)
In conclusion, a set of factors that includes the ability of the malaria parasite to rapidly mutate and build resistance to drugs, the low efficacy of current drugs, combined with the mentioned side effects and shortcomings, indicate that a better solution is needed in order to treat malaria and reduce disease transmission.
Exchange Transfusion
Exchange transfusion is often considered the treatment of last resort for severe and cerebral malaria and it is the only non-drug based anti-malarial treatment currently available. Exchange transfusion is a medical technique used to replace the whole blood or one of its components, e.g., RBCs, of a patient with that of healthy donors. Exchange transfusion as a life-saving procedure has been applied to treat various blood-based diseases other than malaria, including neonatal polycythemia, Rh-induced hemolytic disease of the newborn, severe sickle cell crisis, and toxic effects of certain drugs.
Exchange transfusion (ET) as a treatment to malaria was first reported by Gyr et al. in 1974 (Gyr et al., Schweiz Med Wochensch; 104:1628-30, 1974). Since then, the benefits of ET have been reported in numerous research and clinical reports (Griffith Kevin S. et al., JAMA. 297(20):2264-2277, 2007; Hoontrakoon et al., Tropical Medicine and Internal Health, 3(2):156-161, 1998; White N J, Journal of Infection 39:185-186, 1999; Mordmuller et al., Clinical Infectious Diseases 26:850-2, 1998; Udani et al., IJCCM 7(2), 2003; Boctor F N, Pediatrics 116(4), 2005; Boctor et al., Transfusion, 43:549, 2003; Shanbag P et al., Ann Trop Paediatr. 26(3):199-204, 2006; Mehta et al., J. Commun Dis. 38(2):130-8, 2006; Powell et al., Transfusion Medicine Reviews, 16(3): 239-250, 2002). These studies have shown that exchange transfusion may reduce the morbidity rate and contribute to a faster recovery time.
For example, it has recently been reported that performing exchange transfusion on patients in the severe stage of the disease results in a significant health improvement and rapid reduction of parasitemia. One study, in which blood exchange transfusion was performed on a 23-year old patient with 43% parasitemia, cerebral involvement, fever, and jaundice. Following two exchange transfusion treatments the patient recovered, and the parasitemia level rapidly decreased to 0.01%. In another study (Powell et al., Transfusion Medicine Reviews, 16(3): 239-250, 2002) a sample of 27 patients affected by severe malaria were treated with blood exchange transfusion. The level of survival was 89%, and in 25% of the patients, a prompt neurological improvement was observed. Another study (Hoontrakoon et al., Tropical Medicine and Internal Health, 3(2):156-161, 1998) showed that ET was safe and well tolerated by the patients. It also showed a 20% reduction in mortality when ET was used in conjunction with drugs whereas the mortality rate of those patients that did not received ET was as high as 69%.
The effectiveness of the blood exchange transfusion and the rapid recovery of patients with severe malaria following exchange transfusion have been attributed to various factors (Feldman et al., “Tropical and Parasitic Infections in the Intensive Care Unit”, Springer, 2004). First, by removing the infected RBCs from the circulation the parasite burden is lowered. Second, exchange transfusion also permits the rapid reduction of the antigen load, parasite-derived toxins, and parasite-produced metabolites and toxic mediators released in the blood when RBCs explode. Finally, exchange transfusion in malaria patients results in the replacement of rigid pRBCs and unparasitized RBCs with healthy RBCs that are more deformable, thereby alleviating microcirculatory obstruction.
ET is often considered a last resort method and scarcely performed for its numerous risks, high cost, labor-intensive procedure and lack of sufficient donors to provide safe blood. In fact, whereas the potential benefits of ET are significant, ET also has numerous side effects and substantial risks. Risks and complications include but are not limited to blood clots, changes in blood chemistry, heart and lung problems, infections, shocks due to inadequate blood replacement or blood overflow, and immunological rejection risk associated with injecting large volume of whole blood from several donors, each one with a different antibody system.
In addition to these risks, exchange transfusion is a difficult practice requiring significant medical expertise and is inherently impractical on a large scale because of the large amount of blood it requires. Each exchange transfusion requires up to several units of screened and healthy blood. Blood banks even in developed countries like the U.S. and Western Europe run below optimal quantity. In most of the regions affected by malaria, especially in the sub-Saharan regions, the population is also strained by several other infective diseases, poverty, a lack of specialized centers for blood collection and storage, and famine. It is highly unlikely to imagine that enough healthy blood could be collected to effectively solve the malaria problem on a significant scale. In addition, the World Health Organization (WHO) recognized that malaria contributes indirectly to HIV transmission through transfusions with infected blood to patients with severe malaria, and concluded that is not possible to make any general recommendation since the risks may outweigh the benefits associated with exchanging blood between donor and patient. Unable to give direct guidelines, the WHO leaves to the discretion of individual doctors the decision to perform the blood exchange transfusion as the last life-saving resource available.
These factors contribute ultimately to the reasons for the limited use of ET, and help explain why ET is performed only in those extremely severe episodes that manage to reach well-equipped hospitals where the supply of screened blood is readily available, and cost and labor issues are less significant.
Furthermore, although ET has been performed numerous times in numerous hospitals, a comprehensive clinical trial proving the benefits of ET with a statistically significant margin has not yet been completed. However, the lack of extensive study is to be attributed to the underlying awareness in the medical community that even if ET were to be effective, it would not be a mass-applicable technique because of the costs and prohibitive requirements for fresh blood.
Nevertheless, despite the lack of comprehensive clinical trials, the numerous medical reports and research papers provide evidence of the beneficial effects of ET in malaria treatment. Many experts encourage ET as an adjunct treatment for patients with a parasitemia level greater than 10% and for which drug treatment is failing.
Blood Apheresis Technology and its Limitations
The procedures and devices used in an ET may vary depending on the blood composition that is being transfused. In treating malaria, ET may be performed manually or automatically using various apheresis systems. Manual ET is typically performed by withdrawing the patient's blood in small amounts of about 100 ml through a venous catheter. An equal amount of donor's blood is then injected through a secondary blood vessel, which is often placed in the other arm. The cycle is repeated until the correct volume of blood has been replaced, and the level of parasitemia reduced, by either replacing the entire patient's blood volume or a fraction of it, which varies case by case. Typically during an ET process, several blood volumes are transferred from donors to patient (1 unit of blood volume is typically 500 ml).
In automatic ET processes, clinicians may use various apheresis systems (Bruce et al., “Apheresis: Principles and Practice”, American Association of Blood Banks (AABB), 2003). For example, a patient may be attached to a plasmapheresis or erythropheresis machine that continuously withdraws blood and separates the plasma from the RBCs by centrifugation. In a plasmapheresis, the plasma is enriched with healthy RBCs and circulated back to patient's circulatory systems; in an erythropheresis, the RBCs are added to frozen plasma and injected back into the patient's circulatory systems. An example of the use of these devices is found in Boctor et al. (Boctor F N., Pediatrics, 116(4, 2005), in which the author reported using the COBE spectra apheresis system (COBE Laboratories, Lakewood, Colo.) to remove the patient's RBCs and replace them with donor RBCs.
Despite few technical differences, all current devices using ET achieve separation by centrifugation technology leveraging the density difference between the various blood components.
Magnetic Property of Malaria Parasitized Red Blood Cells
Malaria infected RBCs have magnetic properties that differ significantly from those of healthy RBCs and all other blood's cells (Hackett et al., Biochimica et Biophysica Acta, 1792: 93-99, 2009; Sullivan et al., “Biopolymers, Volume 9”, Wiley-VCH Verlag GmbH & Co, P., 2002; Moore et al., FASEB J. 2006 April; 20(6):747-9. Epub 2006 Feb. 6.). After penetrating the RBCs, the parasite converts the heme groups into an insoluble highly compacted crystal known as “hemozoin”. The conversion is made by the parasite to detoxify the heme. The hemozoin is present in all intra-erythrocyte stages of the parasite—the ring, trophozoite, schinzont, and gametocyte stages—and it occurs in all Plasmodium falciparum species. When the parasite reaches maturity and the RBC bursts, the hemozoin is released in the blood and scavenged by white blood cells.
Each heme-group contains a high-spin Fe+3 (S=5/2) stacked in close proximity. The Fe—Fe atomic separation is around 8 angstrom (Andrzej et al., J. Am. Chem. Soc. 128:4534-4535, 2006). The transformation of low-spin (Fe+2) diamagnetic oxyhemoglobin into high-spin (Fe+3) hemozoin and the close proximity of the Fe atoms give rise to the strong paramagnetic properties of the hemozoin. Studies have shown that the amount of heme converted into hemozoin increases linearly during the parasite intra-erythrocyte lifecycle. It has been estimated that prior to the RBC bursting, nearly 80% of the hemoglobin was consumed and its heme converted to hemozoin.
Hemozoin has been used in various ways—to develop vaccine and drug treatments, diagnostic modality, and for a magnet-based technology to enrich pRBCs. The following is a list of representative patents related to the use of hemozoin: U.S. Pat. Nos. 5,116,965; 5,130,416; 5,296,382; 5,393,523; 5,395,614; 5,476,785; 5,478,741; 5,604,117; 5,827,681; 5,849,307; see also WO89/01785; and WO92/12129.
Because the hemozoin is contained in the parasite vacuole, the parasite itself, and the pRBCs are also paramagnetic. Experimental studies show that pRBCs increase their magnetic susceptibility as they age, because of the increased amount of hemozoin produced by the parasite (Hackett et al., Biochimica et Biophysica Acta, 1792: 93-99, 2009; Moore et al., The FASEB Journal Express Article doi:10.1096/fj.05-5122fje, Published online Feb. 6, 20066). These studies showed that in the late stages, the pRBCs had magnetic susceptibility of between 1-2×10−6 in (SI) unit, which is larger by a factor of about 10-fold than the magnetic susceptibility of un-parasitized oxygenated RBCs and of all other blood cells and plasma. For example, oxygenated un-parasitized RBCs have magnetic susceptibility around −0.2×10−6 in (SI) unit (Coryell et al., Proc. Natl. Acad. Sci. U.S.A. 22(4): 210-216, 1936; Taylor D S, J. Am. Chem. Soc., 60(5), pp 1177-1181, 1938). In these studies magnetic susceptibility is often reported with respect to water, since plasma has magnetic susceptibility very close in magnitude to the water's susceptibility. The magnetic property of the pRBCs has been exploited using a magnetic separator, of the type described in next section, as a technique to enrich pRBCs for routine culture and analysis. For example, early researchers have used magnetic field gradients to remove pRBCs from small blood samples of about 10 ml or less (Paul et al., The Lancet, 318(8237):70-71, 1981). Others have used the magnetic properties to design diagnostic tests. Recently, isolation of malaria pRBCs using commercial high-gradient magnetic separators (MACS technology, Miltenyi Biotec GmbM, Gladbach, Germany) has been performed with levels of separation efficacy close to 95% (Uhlemann et al., Macs&more, 4(2), 2000). These separators are substantially equivalent to the apparatus described in U.S. Pat. Nos. 3,567,026, 3,676,337 and 3,902,994.
Magnetic Cell Separation Technology and its Limitations
A variety of bioparticle isolation and magnetic separation devices are known; see e.g., U.S. Pat. No. 6,361,749. In this section we address limitations of certain of these prior art devices and methods. Separation of pRBCs has been achieved using high-gradient magnetic separation of the type described, e.g., in U.S. Pat. Nos. 3,567,026, 3,676,337, 3,902,994 and 5,691,208. In each such apparatus, a magnetic (steel) wool is placed into the separation chamber directly in contact with the test medium containing the target cells. Target cells that can be separated by this method are typically classified in two categories. The first includes cells that are either permanently magnetized or that have strong paramagnetic or ferromagnetic properties. The second category comprises cells that have very small magnetic properties but can be bound to magnetic beads coated with antibodies which bound specifically to the target cells. The separation chamber is then placed in a magnetic field created by permanent magnets, superconducting magnets, or electromagnets. The function of the steel or iron wool is to create a high magnetic field gradient, which generates a magnetic force on the magnetic particles within the medium, attracting and retaining the magnetic particles. In the typical operation method, the medium is placed into the separation chamber and under the force of gravity it percolates through the steel wool. While percolating, the magnetic labeled cells are retained by the iron or steel wool and in this process the medium is purified. Retention of targeted cells by magnetized steel or iron wool is one of the distinguishing characteristics of these devices. The use of gravitational force as the propulsive method is a second distinguishing characteristic of these apparatus.
The commercially available device MACS (Miltenyi Biotec GmbM, Gladbach, Germany) may be used as a bench top apparatus in routine analysis for research, but is not suited for separating large volumes of blood or for high concentrations of pRBCs. For example, a typical device of this kind with a steel or iron wool matrix of 13 cm3 separates pRBCs from whole blood at a rate of 2 ml/min. Therefore, to purify the average amount of blood in an adult (i.e., about 5-6 liters) using such devices would take over 50 hours of processing time. Furthermore, such devices are subject to clogging when the density of cells to separate is large. Typically a steel or iron wool filter of 40 ml in volume may trap about 1.3*1010 RBCs, after which the device clogs. In the blood of an adult patient with a parasitemia, e.g., of 20% and hematocrit of 40% (typical values for severe malaria) there are about 5*1012 pRBCs. Thus, a device using steel wool as a separating mechanism would require a filter with a volume as large as 16 liters.
There are other classes of devices that use a high magnetic field gradient to generate attractive forces and separate magnetic cells from non-magnetic mediums. See, e.g., U.S. Pat. Nos. 4,663,029, 5,465,849 and 6,688,473. Here, magnetic cells or particles in fluid are enclosed in a chamber and are separated by deviating the particle trajectory accordingly to their magnetic susceptibility. For example, in U.S. Pat. No. 4,663,029, a separator comprising a non-magnetic canister with a single magnetized wire extending parallel to the canister is described. This configuration limits the canister's height to about twice the wire's radius, and the canister's width to the wire diameter. Therefore, the cross section in this apparatus is limited to an area of about 4a^2. For small cells like pRBCs with a susceptibility of about 1-2×10−6 in (SI) unit, in a blood medium, to generate forces comparable in magnitude to the typical viscosity force the cells experience in the chamber (˜10-100 pN), a wire must have radius a ranging between 10-100 μm. An apparatus of the type described in U.S. Pat. No. 4,663,029 applied to the separation of pRBCs would thus require a cross section of about 4×10−8 meter2, which at a fluid velocity of about ½ meter/sec would result in a flow rate lower than 2×10−8 meter3/sec. At this rate, more than 80 hours of processing time would be required to treat 5-6 liters of blood. And, while an apparatus of the type described in U.S. Pat. No. 5,465,849 applied to the separation of pRBCs might remove pRBCs from a patient, such an apparatus would also likely remove large amounts of the medium that surrounds the pRBCs (i.e., blood). As a result, an already weakened patient would be deprived of a portion of their healthy blood components. Moreover, after using an apparatus as described in U.S. Pat. No. 5,465,849, a large volume of blood and biohazard material would be remain as a waste product after the apparatus was used. This large volume of blood would then need to be safely disposed, thereby resulting in higher safety risks and processing costs.
Current magnetic cell separation technology is not applicable to a dialysis-like process where the patient's blood must be processed in short time, in continuous mode, and the pRBCs separated from the healthy RBCs and extracted from the patient blood circulatory system. For this and other reasons, it would be useful to have new and improved devices and methods for separating magnetically reactive materials from blood. Such devices and methods will be useful for treating patients with conditions characterized at least in part by infected or otherwise abnormal blood cells having magnetic properties that differ from normal or uninfected blood cells in the patient.